Glycosyltransferases are enzymes involved in in vivo biosynthesis of sugar chains on glycoproteins, glycolipids and the like (hereinafter referred to as “complex carbohydrates”). Their reaction products, i.e., sugar chains on complex carbohydrates have very important functions in the body. For example, sugar chains have been shown to be important molecules primarily in mammalian cells, which play a role in cell-cell and cell-extracellular matrix signaling and serve as tags for complex carbohydrates during differentiation and/or development.
Erythropoietin, a hormone for blood erythrocyte production, can be presented as an example where sugar chains are applied. Naturally-occurring erythropoietin is disadvantageous in that it has a short-lasting effect. Although erythropoietin is inherently a glycoprotein, further attempts have been made to add new sugar chains onto erythropoietin, as a result of which recombinant erythropoietin proteins with an extended in vivo life span have been developed and produced and are now commercially available. In the future, there will be increasing development of such products in which sugar chains are added or modified, including pharmaceuticals and functional foods. Thus, it is required to develop a means for freely synthesizing and producing sugar chains. In particular, the development of glycosyltransferases is increasing in importance as one of the most efficient means.
Until now, about 150 or more glycosyltransferase genes have been isolated from eukaryotic organisms including humans, mice, rats and yeast. Moreover, these genes have been expressed in host cells such as CHO cells or E. coli cells to produce proteins having glycosyltransferase activity. On the other hand, about 20 to 30 types of glycosyltransferase genes have also been isolated from bacteria which are prokaryotic organisms. Moreover, proteins having glycosyltransferase activity have been expressed in recombinant production systems using E. coli and identified for their substrate specificity and/or various enzymatic properties.
Sialic acid is often located at the nonreducing termini of sugar chains and is therefore regarded as a very important sugar in terms of allowing sugar chains to exert their functions. For this reason, sialyltransferase is one of the most in demand enzymes among glycosyltransferases. As to β-galactoside-α2,6-sialyltransferases and their genes, many reports have been issued for those derived from animals, particularly mammals (Hamamoto, T., et al., Bioorg. Med. Chem., 1, 141-145 (1993); Weinstein, J., et al., J. Biol. Chem., 262, 17735-17743 (1987)). However, such animal-derived enzymes are very expensive because they are difficult to purify and hence cannot be obtained in large amounts. Moreover, such enzymes have a problem in that they have poor stability as enzymes. In contrast, as to bacterial β-galactoside-α2,6-sialyltransferases and their genes, reports have been issued for those isolated from microorganisms belonging to Photobacterium damselae (International Publication No. WO98/38315; U.S. Pat. No. 6,255,094; Yamamoto, T., et al., J. Biochem., 120, 104-110 (1996)).
Various mammalian and bacterial sialyltransferases previously known are reported to have an optimum reaction pH in an acidic range, e.g., between pH 5 and 6 (Paulson, J. C. et al., J. Biol. Chem., 252, 2363-2371 (1977), Yamamoto, T., et al., J. Biochem., 120, 104-110 (1996)). It is widely known that sialic acid attached to simple sugar chains or complex carbohydrate sugar chains on various glycoproteins, glycolipids and the like is gradually degraded under acidic conditions. Moreover, CMP-sialic acid, which is a glycosyl donor substrate of sialyltransferase and is extremely high in price, is known to be rapidly degraded under acidic conditions, but extremely stable under alkaline conditions. Thus, in the case of using sialyltransferase for transfer of sialic acid to various complex carbohydrates or sugar chains, there is a demand for sialyltransferase having an optimum reaction pH in a neutral to alkaline range, in terms of post-reaction stability and storage properties of sialic acid-containing sugar chains and also in terms of efficient use of CMP-sialic acid for use in sialic acid transfer reaction.
Patent Document 1: International Publication No. WO98/38315
Patent Document 2: U.S. Pat. No. 6,255,094
Non-patent Document 1: Hamamoto, T., et al., Bioorg. Med. Chem., 1, 141-145 (1993)
Non-patent Document 2: Weinstein, J., et al., J. Biol. Chem., 262, 17735-17743 (1987)
Non-patent Document 3: Yamamoto, T., et al., J. Biochem., 120, 104-110 (1996)
Non-patent Document 4: Paulson, J. C. et al., J. Biol. Chem., 252, 2363-2371 (1977)